RET Fusion Testing in Patients With NSCLC: The RETING Study

Introduction RET inhibitors with impressive overall response rates are now available for patients with NSCLC, yet the identification of RET fusions remains a difficult challenge. Most guidelines encourage the upfront use of next-generation sequencing (NGS), or alternatively, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) when NGS is not possible or available. Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in patients with advanced NSCLC. Methods This situation prompted us to evaluate several RET assays in a large multicenter cohort of RET fusion–positive NSCLC (n = 38) to obtain real-world data. In addition to RNA-based NGS (the criterion standard method), all positive specimens underwent break-apart RET FISH with two different assays and were also tested by an RT-PCR assay. Results The most common RET partners were KIF5B (78.9%), followed by CCDC6 (15.8%). The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET(12) fusion. The three RET fusions not identified with RT-PCR were AKAP13(35)-RET(12), KIF5B(24)-RET(9) and KIF5B(24)-RET(11). All three false-negative RT-PCR cases were FISH-positive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays. Signet ring cells, psammoma bodies, and pleomorphic features were frequently observed (in 34.2%, 39.5%, and 39.5% of tumors, respectively). Conclusions In-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for the rapid and effective detection of RET fusions in patients with NSCLC. The likelihood of RET false-negative results with both FISH and RT-PCR reinforces the need for upfront NGS in patients with NSCLC.


Introduction
2][3] RET activation occurs when the GDNF ligands bind to their receptors, causing homodimerization, autophosphorylation, and ultimately activation of downstream signaling pathways. 4ncogenic activating fusions have been identified in a variety of malignant tumors, including papillary thyroid carcinomas and NSCLC. 4,5RET fusions are found in 1% to 2% of NSCLC, and there is a higher prevalence in never or light smokers, younger age, and adenocarcinoma (AC). 4,5In treatment-naive patients, RET fusions tend to be mutually exclusive with other major oncogenic drivers. 4The rearrangements typically involve the 3' kinase domain of RET encoded by exons 12 to 18 to various 5' heterologous upstream partner genes. 4In NSCLC, the most typically reported RET partners are KIF5B (w70%), CCDC6 (w20%), and NCOA4 (w2%), and many other partners have been reported as isolated examples. 6][17][18][19][20][21][22][23][24] The available diagnostic methodologies used to identify RET fusions include the increasingly popular next-generation sequencing (NGS) and single-gene approaches such as fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR). 20,25Accordingly, in clinical trials, there is vast heterogeneity in local testing methods, and between 18% to 42% of patients have been identified by either FISH or RT-PCR. 12,15,16,19everal professional organizations and academic groups have released recommendations on the standard methods to detect RET fusions in daily practice and clinical research. 6,10,26,27Most guidelines encourage the upfront use of NGS, or alternatively, FISH or RT-PCR when NGS is not possible or available. 6,10,26Although break-apart FISH has traditionally been the accepted standard test for the detection of fusions, RET FISH is especially difficult to interpret and may be susceptible to both false negatives and false positives. 10Moreover, the real-world performance of specific RT-PCR assays remains largely unknown.
Taken together, the suboptimal performance of single-analyte assays to detect RET fusions, although consistent with the notion of encouraging universal NGS, is currently widening some of the clinical practice gaps in the implementation of predictive biomarkers in advanced NSCLC. 28Therefore, we hypothesized that in-depth knowledge of the advantages and disadvantages of the different RET testing methodologies could help clinical and molecular tumor boards implement and maintain sensible algorithms for rapid and effective detection of predictive biomarkers (i.e., including RET) in patients with NSCLC.This situation prompted us to evaluate several RET assays (i.e., RNA-based NGS as criterion standard method, FISH, and RT-PCR) in a large multicenter cohort of RET-positive NSCLC to obtain real-world data.

Study Design and Tumor Samples
The flow diagram is depicted in Figure 1.There were 57 RET fusion-positive samples from patients with NSCLC that had been initially tested as part of routine clinical care in 16 different institutions, were used for this study (also known as RETING or RET and Individual gene assays & Next-Generation sequencing).To confirm the RET fusion-positive status, targeted RNA-based NGS analysis (the criterion standard method) was performed at the referral institution.Only cases with enough tissue available (i.e., a minimum of 20% tumor cell content) were included.In addition, 100 consecutive RET NGSnegative samples from NSCLC tested at the referral institution as part of routine clinical care were included as negative controls.The material available for all tumors was formalin-fixed and paraffin-embedded (FFPE).The specifics of formalin-fixation were unknown.All cases were reviewed by three pathologists (E.C., F.L.R., and J.L.R.C.).In addition to NGS, all positive specimens underwent break-apart RET FISH with two different assays using an automated scanning system and were also tested by an RNA PCR-based assay.In the negative cohort, only one RET FISH assay was investigated.The Institutional Ethics Committee at Fundacion de Investigation HM Hospitales and Hospital Universitario 12 de Octubre reviewed and approved this study.Each referring institution regulated the need for additional specific consent.Clinical data from the RET NGS-positive cohort were retrieved from the patient clinical records.

NGS for RET Fusions
A targeted RNA-based NGS panel (Oncomine Comprehensive Assay v3 test [ThermoFisher Scientific, Waltham, MA]) was performed for all cases (positive and negative) on the Ion S5 sequencer with automated library preparation using the Ion Chef System, as described previously. 29For each FFPE tumor sample, freshly cut 5-mm-thick sections were collected on separate Eppendorf tubes for DNA and RNA extraction: three sections for surgical specimens and five sections for small biopsy specimens for each tube.The first and last sections were stained with hematoxylin-eosin and reviewed by two pathologists (E.C. and F.L-R.) to confirm that the percentage of tumor cells was greater than or equal to 20%.The DNA extraction was performed with the Cobas DNA Sample Preparation Kit (Roche Molecular Systems, Pleasanton, CA) following the manufacture rs instructions.The RNA extraction was performed with the High Pure FFPET RNA Isolation Kit (Roche Molecular Systems) following the manufacturer's instructions.The RNA was then purified and concentrated by using the GeneJET RNA cleanup and concentration micro kit (ThermoFisher Scientific).The protocol for the NGS analyses followed the manufacture rs instructions, and a minimum of 500,000 mapped fusion panel reads was required for RET fusion analysis.The RET NGS result was used as the criterion standard method and the complete NGS report was only available for the RET NGSpositive cohort.

FISH for RET fusions
FISH was carried out on unstained 4-mm-thick FFPE tumor tissue sections from all cases.For all positive cases, we used two commercial break-apart RET FISH assays: Vysis RET FISH Break-Apart Probe RUO kit (Abbott Molecular, IL) and ZytoLight SPEC RET Dual Color BreakApart Probe (ZytoVision GMbH, Bremerhaven, Germany).In the negative cohort, we only investigated the Vysis RET FISH probe.The methodologies have been described in detail elsewhere. 30,31RET FISH assays were independently captured and scored with the automated BioView Duet scanning system (BioView, Rehovot, Israel) by a thoracic pathologist (E.C.) or molecular biologist (S.H.).A minimum of 50 tumor nuclei were counted.RET FISH-positive cases were defined as those with greater than or equal to 15% break-apart signals (separated by more than one signal diameter) or isolated 3' signals in tumor cells. 26,32,33sing our own prespecified criteria, if the separation between the signals was greater than one but less than two signal diameters, the pattern was named "borderline positive break-apart."RET FISH-negative samples were defined as those with fusion signals, isolated 5' signals, or less than 15% of positive cells. 26,32,33

Results
The clinicopathologic characteristics of patients with RET fusions are presented in Table 1.

RET Fusions Assessed by FISH
All 138 specimens (positive and negative) were successfully tested by FISH (Fig. 1).In agreement with the NGS results, 36 out of the 38 (94.7%)RET NGS-positive samples were RET FISH-positive by both probes.The overall results were very similar for both probes.The mean percentage of positive cells was 74.6% (median 77%, range 16%-100%) using the Vysis probe and 70.5% (median 74%, range 18%-96%) with the Zyto-Vision probe.The break-apart pattern was more frequently observed than the isolated 3' signal pattern (30 of 36, 83.3% versus 6 of 36, 16.7%) (Fig. 2A-D).The number of cases with a borderline positive break-apart pattern (see definition above) was higher with one of the probes (13 of 36, 36.1% for ZytoVision versus 6 of 36, 16.7% for Vysis).Interestingly, this borderline pattern was identified in all fusion partners except AKAP13.The frequencies were higher for CCDC6 (3 of 6, 50% with ZytoVision and 1 of 6, 16.7% with Vysis) than for KIF5B (9 of 28, 32.1% with ZytoVision and 4 of 28, 14.3% with Vysis) (Fig. 3A).The two RET NGS-positive but FISH-negative samples contained a KIF5B(15)-RET( 12) fusion (Fig. 3B).Both ACs exhibited psammoma bodies and were diagnosed in a surgical specimen.These two patients received a RET TKI and had partial responses.The FISH results for all cases from the negative cohort agreed with those obtained by NGS (Fig. 3C).

RET Fusions Assessed by RT-PCR
Three RET NGS-positive cases were excluded for lack of tumor tissue after the previous analyses (Fig. 1).There were 32 out of the remaining 35 (32 of 35, 91.4%) NGS-positive samples that were RT-PCR-positive.The three RET fusions not identified with RT-PCR were AKAP13(35)-RET (12), KIF5B(24)-RET(9) and KIF5B(24)-RET (11).All three cases were AC that were diagnosed by surgical specimens (n ¼ 2) or core-needle biopsy (n ¼ 1).Both surgical specimens contained either signet ring cells or psammoma bodies.All three cases were FISHpositive, exhibited a typical break-apart pattern, and contained a very high number of positive tumor cells with both FISH assays (82%, 92%, and 90% for Vysis; 60%, 94%, and 96% for ZytoVision, respectively).Of note, two of these three patients received a RET TKI and had partial responses.

Discussion
The information presented herein is very timely because a recent survey from more than 500,000 patients has identified that almost 50% of patients with advanced NSCLC were not candidates for targeted therapies because of biomarker testing issues. 28The clinical gaps can be summarized as follows: tissue (insufficient tissue or inaccurate estimation of tumor cell content), testing (appropriate assay was not ordered or results were inconclusive or false-negative), and time (turnaround time delays). 28Therefore, in some series the frequency of RET fusions falls below 1% 11,[34][35][36] and, unsurprisingly the percentage is within the expected range (i.e., 1%-2%) in fully genotyped cohorts. 360][41][42] Until NGS is routinely performed in all patients with advanced NSCLC, a deep understanding of the concept of "molecular redundancy" is reassuring. 43This notion has been recommended and endorsed by all the major professional organizations in the field and can be summarized as follows: "Laboratories should ensure that test results that are unexpected, discordant, equivocal, or otherwise of low confidence are confirmed or resolved using an alternative method or sample." 43Therefore, in this RETING study, we wanted to explore the performance of typically used single-gene RET assays as potential complementary tools to NGS in testing workflows for patients with advanced NSCLC. 6,10,26easoning that RNA sequencing is now becoming the accepted standard for fusion identification, because of its superior sensitivity, 40,44 we decided to use as our standard criterion a large RNA-based NGS assay that required very little input RNA.The molecular landscape of RET fusions in our series is remarkably similar to previous reports (i.e., high frequency of co-occurring TP53, SETD2, and CTNNB1 mutations), 7,8,10,[45][46][47][48] including the puzzling finding of MDM2 and CDK4/6 amplifications. 8,46[45][46][47][49][50][51][52][53][54] The most common fusion partners are KIF5B, CCDC6 and NCOA4.Several conclusions can be drawn from our study.First, the performance of two typically used FISH probes was similar: two clear-cut false-negative results with both probes on the same samples.It is unfortunate that both suboptimal readings involved the most frequent RET fusion in patients with NSCLC (i.e., KIF5B [15]-RET [12]  fusion) (Table 2).Despite the initial description that RET FISH false-negative results were restricted to the NCOA4 partner, 10 isolated examples involving KIF5B fusions have been reported. 33,46,47,55Second, our absence of RET FISH false-positive results could be because of the use of an outstanding automated FISH scanning system and a large NGS panel as a FISH comparator.In agreement with other authors, we believe that the current falsepositive rate of RET FISH could be overestimated for two main reasons: (1) the adoption of a low threshold of signal separation for positive break-apart signals or a low percentage of positive nuclei as the cutoff for positivity, 56 and (2) the use of RT-PCR or small NGS panels as a standard criterion, which may miss some fusion partners. 33,53,57Moreover, similarly to other break-apart FISH probes, 58 the presence of complex patterns in RET FISH assays (e.g., loss of signals) is clearly linked to false-  positive results. 10,32,338,53,59-63 When using RT-PCR it is important to understand the concept of "diagnostic sensitivity," which relates to the comprehensiveness of the assay, or the percentage of all RET fusions described for the gene detectable by the given assay. 6,64Users of these assays should be constantly aware that "pseudo false-negatives" (i.e., because fusion partners are not included in the design of an assay) are unavoidable.Accordingly, three RET fusions were missed by the RT-PCR kit, emphasizing the need to always consider NGS testing in patients with driver-negative NSCLC. 6A review of the literature in light of our findings suggests that the presence of an AKAP13 partner is a rare event.Unfortunately, the lack of detail regarding the specific KIF5B breakpoints in some large series prevents drawing definitive conclusions regarding the molecular epidemiology of KIF5B(24)-RET(9) and KIF5B(24)-RET( 11) fusions (Table 2).According to Mizukami et al., 65 the frequency of the KIF5B(24)-RET (11) fusion across several cohorts comprising 60 patients is 2%, which is similar to our experience (2.6%).Nevertheless, the occasional presence of this fusion in two very small series (13 and 14 patients with a frequency of around 7%) remains worrisome and highlights the difficulty in calculating the risk of false-negative results when using RT-PCR for RET testing. 59,62Single-analyte assays are still very popular across the globe for cost reasons or because exclusionary testing is implemented in high EGFR mutation prevalence regions. 6In exclusionary testing, several biomarkers are tested first, followed by NGS in drivernegative patients.Despite contradictory reports on the cost-effectiveness of this strategy, [66][67][68] recently released expert consensus or recommendations from the Asia-Pacific region support the use of upfront NGS in patients with NSCLC. 27,69lthough RET immunohistochemistry to detect RET fusions is not currently recommended because of its wide range of sensitivity (50%-100%) and specificity (30%-90%), 6,10,26,27 several comments might be helpful for the future implementation and development of RET antibodies: (1) evidence on the topic is still inconclusive because of the small sample size of many reports and the insufficient representation of non-KIF5B partners 26 ; (2) only antibodies directed to the C-terminal portion of RET should be used to identify the chimeric protein 26 ; and (3) the clone EPR2871 is probably the most frequently used and well characterized, with an interesting association between the fusion partner and the expression of the protein. 10Some authors have reported higher H-scores for KIF5B fusions, which resulted in perfect sensitivity for the detection of KIF5B-RET fusions. 10he histologic characteristics of our NSCLC with RET fusions is concordant with the literature.[51][52][53]62,63 That RET fusion-positive AC can contain signet ring cells (range: 27-36%, mean: 30.7%, median: 30%) and psammoma bodies are well known, 62,70 but the predictive value of these features is not fully recognized in clinical practice.Of note, four of the five false-negative FISH/RT-PCR samples contained either signet ring cells or psammoma bodies.Accordingly, pathologists should always report them and persevere in the search for actionable fusions in those circumstances, as they can also be found in NSCLC with ALK or ROS1 fusions. 30,31,70Another interesting and underrecognized feature is the presence of papillary or micropapillary patterns in RET fusion-positive lung AC: almost 9% of the AC in the present series exhibited either one and reported rates to range from 9% to 36% (median: 20%, media: 22%). 48,62,63In agreement with other authors, both KIF5B and non-KIF5B partners were involved in papillary formation. 62,63,712,72,74 Interestingly, neuroendocrine differentiation can also be found in pancancer studies of RET fusion-positive solid tumors, highlighting the need to also use histologic classification as a way to increase the likelihood of finding an actionable fusion in tumoragnostic approaches, as counterintuitive as it might seem. 21,22,29,49n conclusion, the potential for false-negative results with single-analyte assays reinforces the need for upfront NGS in patients with NSCLC.A consideration of the clinical problem of NSCLC highlights the need to be aware of how the methods that we use perform in the real-world setting.BA, break-apart; NSCLC, non-small cell lung carcinoma.

April 2024
The RETING Study

Figure 2 .
Figure 2. Representative examples of RET FISH-positive NSCLCs using the Vysis RET Probe (A,B) and the ZytoVision RET Probe (C,D).(A,C) A typical break-apart pattern is shown with one fused signal and one break-apart signal per nucleus (arrows).(B,D) An isolated 3' signal pattern is depicted (red signals with the Vysis probe and green signals with the ZytoVision probe) (arrows).All four cases were scored using the BioView Duet scoring system and were RET NGS-positive.See text for details.Original magnification: x1000.FISH, fluorescence in situ hybridization; NGS, next-generation sequencing.

Figure 3 .
Figure 3. Representative examples of RET FISH patterns: borderline break-apart positive (A), false-negative (B), and (C) typical negative fusion signal pattern (C).(A) A tumor with a CCDC6-RET fusion showing a borderline break-apart positive pattern.(B) A tumor with a KIF5B-RET fusion showing insufficient separation between the red and the green signals (i.e., FISH false-negative).(C) A typical example of a tumor without RET fusions exhibits two fused signals.All images correspond to the Vysis RET probe and were interpreted using the BioView Duet scoring system.The fusion status was confirmed by NGS.See text for details.Original magnification: x1000.FISH, fluorescence in situ hybridization.

a
Only series with genomic confirmation of more than 15 cases and reproducible FISH protocols are included.bDespite of the lack of genomic confirmation this series is included due to the high quality of the FISH data.

Table 1 .
Clinicopathologic Features of Patients with RET Fusions a Clinical information was available for 37 patients.bPatientswith stage IV disease (n¼28).cPatientswith stage IV disease treated with RET TKI therapy (n¼21).AC, adenocarcinoma; NSCLC-NOS, non-small cell lung carcinoma, not otherwise specified; TKI, tyrosine kinase inhibitor.

Table 2 .
Summary of Studies Addressing the Tissue Detection Rate of RET Fusions in Patients with NSCLC a a Only studies with more than 20 RET-positive cases are included.bThedenominatoristhe total number of RET fusions.cCorresponds to a AKAP13(35)-RET(11).dThespecific breakpoint is only available for 16 of the 22 RET fusions.NSCLC, non-small cell lung carcinoma.

Table 3 .
Summary of Studies Addressing the use of FISH to Detect RET Fusions in Patients with NSCLC a Study 10ng et al.10